The microfluidic technology is a biochemical analysis technology that has been developed rapidly in recent years. It has potential advantages such as high throughput, high detection sensitivity, low cost and easy automation, making it have a wide application prospect in the field of biochemical detection, especially in the field of biomedicine.
Droplet microfluidic technology is an important component of microfluidic technology. The micro-droplet microfluidic technology is to transfer two mutually incompatible fluids, taking the most common water and oil as an example, into the micron-scale channel, so that the water phase is divided into small micro-droplets of stable size, on the order of micrometers by the oil phase through the action of fluid mechanics Each micro-droplet acting as an independent reactor, is equivalent to the tube commonly used in biochemical reactions. The “small tube” of micro-droplets is small in size and large in number, and has many advantages such as high throughput, low reagent consumption and low background noise, so it has a good industrialization prospect.
The high throughput of micro-droplet technology benefits from the large number of micro-droplets and small volume, but it also has a challenge for detecting micro-droplet fluorescence signals. If the number of micro-droplets is large, the detection rate of the micro-droplet fluorescence is required to be high, otherwise high throughput cannot be achieved. The small volume of the micro-droplets has a challenge to the intensive reading of the detection system. At present, the micro-droplet fluorescence detection methods used in papers and commercial instruments mainly include the following three methods.
The first method is to separate the micro-droplets by the sheath flow and sequentially pass through the detection zone containing the excitation light and the detection light path, and then systematically record the fluorescence intensity of each micro-droplet. This detection method is similar to flow cytometry, which is characterized in that an optical path system is fixed and a sample passes through the detection zone in turn. This type of detection is widely used not only in academia, but also in existing micro-droplet commercial instruments, including Bio-Rad's QX200 and RainDrop Digital PCR System from Raindance Technologies in the USA. This detection method has the advantages of high precision, high sensitivity and good stability of micro-droplet fluorescence detection. The disadvantage is that there is a bottleneck in the micro-droplet detection rate, since the micro-droplet flow velocity is limited by its stability. Raindance's RainDrop Digital PCR System takes about half an hour to read the fluorescence of about 5 million micro-droplets, and detects about 3,000 micro-droplets per second. Such detection rate can not meet the detection requirements of clinical high-throughput detection.
The second method is to use fluorescence imaging to capture a certain number of micro-droplet fluorescence images at a time, and then use image processing technology to automatically identify the micro-droplet fluorescence in the image to obtain the fluorescence information of the micro-droplets. Due to the large imaging range, this detection method does not require sample flow during detection and requires less fluid driving system; and it can detect not only micro-droplets in microchannels, but also on chip cavities and even slides. The requirement on the detection environment where the microdroplets are located is low. The defect is that imaging picture is required for image processing, and image processing is not only complicated, but also computationally intensive, requiring high hardware and software support; in addition, because of the need to use a camera to acquire images, the resolution capability for the intensity of the fluorescence signal is lower than that of the photoelectric multiplier tube, so that the detection of the fluorescence of the droplets is also influenced.
The third method of detecting droplets is to place the droplets in a cylindrical transparent container. The container rotates at a high speed with the central axis of the cylinder as a rotary shaft. Due to the centrifugal force, droplets will be distributed inside the container wall, and the fluorescence is excited. The focus of an optical path coincides with the focus of a fluorescent receiving optical path and is located inside the container wall, then the container rotates by a circle, the fluorescent detection light path can obtain the fluorescence information of the droplets on the “circular ring” corresponding to the position where the focal point is located, the height of the container is changed, and the fluorescent information of droplets on the other “circular ring” can be obtained, the fluorescent signals of all droplets are obtained through the method. This type of detection is in a non-flowing state because the micro-droplets are relatively stationary relative to the container during the detection process, so that there is no micro-droplet stability which affects high-speed movement, achieving a high detection rate, which can be detected approximately 100,000 micro-droplets per second. However, the shortcoming is that the microfluidic technology is not used, the whole detection environment is in an open state, which is easy to pollute the biochemical detection environment and affect subsequent detection. At the same time, the excitation position of each micro-droplet fluorescence is not uniform, and the fluorescent signals cannot be quantified and compared, and the obtained data quality is influenced.